The present invention provides hahella chejuensis-derived recombinant cellulase and a use thereof. The recombinant cellulase according to the present invention has a high stability at a high temperature and an optimized activity at a neutral pH, and therefore has a high availability in a process. Also, since the cellulase according to the present invention has an exoglucanase activity as well as an endoglucanase activity, a monosaccharide can be produced during decomposition of cellulose and an additional treatment required in a fermentation process is not needed, and therefore a cost may be reduced. Such an enzyme activity is advantageous to a fermentation or thermal treatment condition for producing a biofuel, and therefore industrial profits may be achieved.
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1. A recombinant cellulase produced by removing a signal sequence and two cbm6 domains from a hahella chejuensis-derived cellulase (HcCel5H).
2. The recombinant cellulase of
3. The recombinant cellulase of
9. A method of producing a cellulase, comprising:
isolating and purifying the recombinant cellulase from a cell culture of the transformant of
10. A method of decomposing cellulose, comprising:
reacting the cellulase of
11. The method of
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This application is the U.S. National Phase of International Application No. PCT/KR2014/003665, filed Apr. 25, 2014, which claims the priority of Korean Application Nos. 10-2013-0046423, filed Apr. 26, 2013 and 10-2014-0050018, filed Apr. 25, 2014, the disclosures of which are all incorporated herein by reference.
The present invention relates to a Hahella chejuensis-derived recombinant cellulase and a use thereof.
To solve problems of instability of an oil price due to a limited crude oil resource and global warming, the world has focused on developing new alternative energy. Accordingly, biofuels including bioethanol capable of being produced from a regenerable plant resource have greatly attracted attention.
A regenerable plant resource, biomass, mostly consists of a lignocellulose type. Here, the lignocellulose mainly consists of cellulose and hemicellulose as a carbohydrate, and lignin. Among these, the cellulose is hydrolyzed with a cellulase, thereby obtaining a sugar such as glucose which can be easily fermented by a microorganism, and the sugar will be converted into a biofuel through fermentation later.
However, the lignocellulose is difficult to be saccharified because, unlike a sucrose-based or starch-based biomass, cellulose is surrounded by hemicellulose and lignin. Generally, a cellulose hydrolysis conversion by cellulases is about 20% of a theoretical maximum. For this reason, it is necessary to perform a physicochemical pretreatment, and pretreated lignocellulose still needs a large amount of the cellulase. Therefore, to achieve an efficient saccharification of pretreated lignocellulose, it is important to increase an activity of the cellulase. Particularly, since the pretreatment is performed at a high temperature, it is very important to ensure a cellulase having stability even at the high temperature.
While cellulases having an increased activity have been disclosed in Korean Patent Nos. 10-1218341, 10-1137020, and 10-0423670, a cellulase having a high activity and excellent processivity at a high temperature has not been reported yet.
The present invention is directed to providing a recombinant cellulase having a high activity even at a high temperature and excellent processivity.
The present invention is also directed to providing a method of decomposing cellulose with the recombinant cellulase.
To achieve the above-describe objective, the present invention provides a Hahella chejuensis-derived recombinant cellulase from which a signal sequence and two CBM6 domains are removed.
The recombinant cellulase of the present invention may include a sequence for facilitating isolation and purification at a terminal. For example, the recombinant cellulase of the present invention may further include a histidine tag at a terminal of a cellulase of Hahella chejuensis from which a signal sequence and two CBM6 domains are removed, but the present invention is not limited thereto.
In one exemplary embodiment of the present invention, a Hahella chejuensis-derived recombinant cellulase having the amino acid sequence of SEQ ID NO: 1 in the accompanying sequence listing is provided.
The present invention also provides a gene encoding the recombinant cellulase.
In one exemplary embodiment, the gene may include the nucleic acid sequence set forth in SEQ ID NO: 2.
The present invention also provides a recombinant vector expressing the recombinant cellulase.
The present invention also provides a transformant transformed with the recombinant vector.
The present invention also provides a method of producing a cellulase including isolating and purifying a recombinant cellulase from a cell culture of the transformant.
The present invention also provides a composition for decomposing cellulose, which includes the recombinant cellulase.
The present invention also provides a method of decomposing cellulose, which includes reacting the recombinant cellulase with cellulose.
The cellulase of the present invention has a high stability at a high temperature and an optimized activity at a neutral pH, and therefore has a high availability in a process. Also, since the cellulase according to the present invention has an exoglucanase activity as well as an endoglucanase activity, a monosaccharide can be produced during decomposition of cellulose. Thus, an additional saccharification treatment required for a fermentation process is not needed, which reduce the process cost. Such enzymatic characteristics are beneficial for fermentation or thermal treatment condition for producing a biofuel, and industrial application can be achieved.
The present invention provides a Hahella chejuensis-derived recombinant cellulase.
The recombinant cellulase of the present invention is a recombinant cellulase prepared by removing a signal sequence and two CBM6 domains from a Hahella chejuensis-derived cellulase (HcCel5H).
The recombinant cellulase of the present invention may include a sequence for facilitating isolation and purification at a terminal. For example, the recombinant cellulase of the present invention may include an additional histidine tag at a terminal of the Hahella chejuensis-derived cellulase from which the signal sequence and the two CBM6 domains are removed, but the present invention is not limited thereto.
In one exemplary embodiment, the cellulase may have the amino acid sequence set forth in SEQ ID NO: 1, and all mutants having a desired activity of the present invention obtained by one or more mutations such as substitution, deletion, inversion, and translocation with respect to the corresponding amino acid sequence may also be included in the enzyme of the present invention.
According to an exemplary embodiment, the molecular weight of the cellulase may be about 34 kDa, which is analyzed by SDS-PAGE.
Q2SFD8 (Gene ID: 3842516), which corresponds to the cellulase of Hahella chejuensis used as the origin of the recombinant cellulase of the present invention, is originally annotated as an endoglucanase. The protein consists of three main domains, which are a cellulase section and two members of cellulose binding module (CBM) family 6. The recombinant cellulase according to the present invention includes neither a signal sequence nor a CBM6 domain, is isolated and purified through affinity chromatography, and the recombinant cellulose was confirmed as having no difference in activity with respect to substrate or an even higher activity than the original cellulase of Hahella chejuensis.
Meanwhile, a test for measuring an activity with respect to temperature and pH was performed on the recombinant cellulase using soluble cellulose, which was carboxy methyl cellulose (CMC), and thereby it was confirmed that optimal activity was shown at pH 6 and 50° C. Also, as the recombinant cellulase was incubated at different temperatures for a certain time and examined whether its activity was destroyed, it was confirmed that a protein kept its activity even at a high temperature (60° C.).
Also, reaction products produced by the reaction with insoluble cellulose, Avicel, were found to be only monosaccharides such as glucose, and disaccharides such as cellobiose, which was confirmed by TLC and HPLC. This shows that the recombinant cellulase according to the present invention has both activities as an endoglucanase and an exoglucanase. Due to such a property, the recombinant cellulase of the present invention may decompose a cellotriose or higher oligosaccharide, for example, cellotriose, cellotetraose, cellopentaose, or cellohexaose.
The recombinant cellulase of the present invention may be produced by a method of synthesizing a peptide, which is known in the art. For example, using a gene cloning and protein expression system or a peptide synthesizer, the recombinant cellulase of the present invention may be produced by an in vitro synthesizing method.
The present invention also provides a gene encoding the recombinant cellulase.
The gene may have the nucleic acid sequence of SEQ ID NO: 2, and in consideration of degeneracy of a genetic code and the mutant protein as described above, a gene having the base sequence of SEQ ID NO: 2 and a homology of 80% or more, specifically, 85% or more may also be included in the scope of the present invention.
The present invention also relates to a recombinant vector expressing the cellulase.
The term “recombinant vector” used herein is a vector capable of expressing a target protein in suitable host cells, and refers to a gene construct having essential regulatory factors operably linked to express a gene insert.
The vector may include, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage or a virus vector. A suitable expression vector includes a signal sequence or leader sequence for membrane targeting or secretion as well as expression regulatory elements such as a promoter, an operator, a start codon, a termination codon, a polyadenylation signal and an enhancer, and may be constructed in various forms depending on purpose. The promoter of a vector may be constitutive or inducible. Also, the expression vector includes a selective marker for selecting host cells containing a vector, and if it is a replicable expression vector, includes the origin of replication.
The recombinant vector of the present invention may be constructed by inserting a nucleic acid encoding the cellulase into a general vector for expressing an E. coli strain, preferably, a pET21 vector. In one exemplary embodiment of the present invention, as the E. coli expression vector, pET21 was used, but the present invention is not limited thereto, and therefore all of the generally used expression vectors for an E. coli strain can be used without limitation. In one exemplary embodiment of the present invention, the recombinant vector may be constructed by inserting a DNA fragment including the cellulase-encoding gene (SEQ ID NO: 2) of the present invention using the expression vector for an E. coli strain, which is the pET21 vector.
The present invention also relates to a transformant transformed with the recombinant vector.
The transformation includes any method for introducing a nucleic acid into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique depending on a host cell as known in the art. Such a method may be, but is not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring with silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextrane sulfate, lipofectamine, etc.
Also, since an expression level and modification of a protein may vary depending on a host cell, a most suitable host cell may be selected depending on purpose.
The host cells may be, but are not limited to, prokaryotic host cells derived from Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. The host cells may also be lower eukaryotic cells such as a fungi (for example, Aspergillus) or a yeast (for example, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa), or higher eukaryotic cells such as plant cells.
The transformant may be easily produced by introducing the recombinant vector into arbitrary host cells. According to an exemplary embodiment of the present invention, the transformant may be produced by introducing the recombinant vector pET21 expressing a cellulase into the E. coli strain BL21 (DE3).
The present invention also relates to a method of producing a cellulase, which includes isolating and purifying the recombinant cellulase from a cell culture of the transformant.
The recombinant cellulase is preferably purified after the transformant is cultured by a conventional culturing method. The cellulase may be modified at a part of its amino acid sequence in any degree without an influence on cellulose decomposability depending on the base sequence of the insert introduced into the recombinant vector, that is, the encoding gene. The modification includes modification by deletion, insertion or substitution.
The present invention also relates to a reaction composition for decomposing cellulose including the recombinant cellulase.
Since the recombinant cellulase according to the present invention may digest cellulose in an endo or exo type, a monosaccharide can be formed. Accordingly, different from the conventional endoglucanase, which cannot degrade the cellulose into monomeric sugar, the use of recombinant cellulase in the present invention does not require an additional enzymatic saccharification process, contributing to the reduction in the process time and cost.
The present invention also relates to a method of decomposing cellulose, which includes reacting the recombinant cellulase with cellulose.
The reaction may be performed at pH 5 to 7 and 20 to 70° C., for example, 30 to 60° C., and specifically, pH 5.5 to 6.5 and 45 to 55° C., but the present invention is not particularly limited thereto.
Hereinafter, the present invention will be described in further detail with reference to examples according to the present invention, but the scope of the present invention is not limited to the following examples.
Truncated HcCel5H (the recombinant cellulase according to the present invention) was prepared by removing a CBM6 domain from a Q2SFD8 (Gene ID: 3842516)-encoding base sequence (Hch03914) classified as an endoglucanase, which is a cellulase of Hahella chejuensis (
A process of producing the recombinant cellulase according to the present invention will be described in further detail.
A genomic DNA of Hahella chejuensis KCTC 2396 was provided from Professor Kim Ji-hyun at Yonsei University (at that time, a doctor at the Korea Research Institute of Bioscience and Biotechnology). The genomic DNA was used in cloning as a template. As a cloning and protein expression host, Escherichia coli BL21 (DE3) was used.
The Q2SFD8 consisted of three main domains as described above, and for the experiment, primers were designed to produce two types of recombinant proteins: a protein not having a CBM6 domain sequence and a protein having a full domain sequence. Since the upstream region of the base sequence was shared, the same primer (F) was used, and a histidine (6×) sequence was attached to the downstream region of the base sequence to be isolated and purified with Histrap. Information on the primers is as follows.
Primers F and R1 were used for the cloning of a CBM6-removed protein, and primers F and R2 were used for the cloning of a protein having a full sequence. Restriction sites for BamHI (GGATCC) and NotI (GCGGCCGC) were added at the upstream and downstream regions, respectively.
Primer F:
(SEQ ID NO: 3)
CCGGATCCATGCACGCTGTGCCGCCGTTATCG
Primer R1:
(SEQ ID NO: 4)
GGGCGGCCGCTCAGTGATGATGGTGATGGTGAGGAATGGGGTCGGAGCT
TTGGATAATG
Primer R2:
(SEQ ID NO: 5)
GGGCGGCCGCTCAGTGATGATGGTGATGGTGTTTTTGTCCTTTAGTGAT
CTTGAACCAGTTCAGG
A PCR product was obtained using the primers prepared as described above and the template provided previously, and the fragment and a vector pET21a were digested with BamHI+NotI. The resultant products were subjected to gel elution, thereby obtaining fragments, and the fragments were ligased with a T4 ligase and then cloned with plasmids such as pET-HchCore and pET-HchFull, respectively.
The recombinant plasmid was used to transform E. coli BL21 (DE3). The transformed E. coli was cultured in 2 L of a Luria-Bertani broth and grown at 37° C. and 200 rpm until an optical density of 600 nm reached 0.8, at which the temperature of culture medium was cooled to 16° C. Subsequently, the expression was induced with 0.5 mM IPTG Afterward, the resultant E. coli was further cultured for 16 hours and recovered to obtain only cells, and then the cells were mixed in a 20 mM Tris-Cl (pH 8.0) buffer to have a final volume of 40 ml. After a protease inhibitor, 1 mM PMSF, was added to the cells, the cells were disrupted by a sonic generator for 10 minutes. The resultant product was centrifuged again (16,000 rpm, 60 min) to remove a supernatant, which was then filtered with 0.45 nm PVDF, and loaded on a 5 ml Histrap (GE healthcare, USA). Afterward, the column was washed with a 20 mM Tris-Cl (pH 8.0) buffer in which 10 mM imidazole was dissolved to remove an unnecessary protein, and a 100 mM imidazole-added buffer was added to the above-described composition, thereby eluting the recombinant proteins. The purified proteins were separated by 10% SDS-PAGE at 180 V for 60 minutes to collect a high purity protein section, and then concentration and buffer exchange of the protein were carried out using Vivaspin 10000 (Satorious, USA). Afterward, the recombinant cellulase according to the present invention was obtained by loading the resultant protein on a SDS-PAGE gel again. A cellulase (original HcCel5H) having a total sequence, not a truncated form (recombinant HcCel5H), was also isolated and produced by the same process (
To compare the activities of original HcCel5H and recombinant HcCel5H, as a substrates, 1% CMC and 10% Avicel were used as soluble and insoluble celluloses, respectively, and the enzymatic reaction were performed in a total volume of 200 μl and at 50° C. Reaction times for CMC and Avicel were 20 minutes and 24 hours. Truncated HcCel5H and original HcCel5H were added at 0.1 mmol per g cellulose. The reaction mixture was boiled at 95° C. for 5 minutes to terminate the enzymatic activity, and the sugars produced from cellulose were quantified by DNS method.
To determine the optimum pH of the recombinant HcCel5H prepared in Example 1, a CMCase activity of the recombinant HcCel5H depending on pH was measured using 1% CMC as a substrate in a total reaction amount of 200 μl for a reaction time of 20 minutes at a reaction temperature of 50° C. At pH 3 to 6, a sodium citrate buffer was used, at pH 7 to 8, a Tris-Cl buffer was used, and at pH 9, a glycerol-NaOH buffer was used. When the reaction was finished, the activity of the enzyme was blocked by boiling at 95° C. for 5 minutes, and quantified by a DNS method.
To determine the optimum temperature of the recombinant HcCel5H prepared in Experimental Example 1, a temperature was measured using 1% CMC as a soluble substrate in a total reaction amount of 200 μl for a reaction time of 20 minutes at pH 6 in a 0.1M sodium citrate buffer. When the reaction was finished, the activity of the enzyme was blocked at 95° C. for 5 minutes, and quantified by a DNS method.
Activities were measured by temperature using insoluble cellulose, unlike Experimental Example 3. The activity was measured using an insoluble substrate, which was 10% Avicel, in a total reaction amount of 200 μl for a reaction time of 12 hours at pH 6 in a 0.1M sodium citrate buffer. When the reaction was finished, the activity of the enzyme was blocked by boiling at 95° C. for 5 minutes, and quantified by a DNS method.
The amount of a protein was measured by Bradford analysis after incubation in different temperatures for 1 and 14 hours at pH 6 (0.1M sodium citrate). The protein stability was assessed by measuring the initial protein amount using Bradford assay, which was compared to the protein amount after incubation for a predetermined time at a varying temperature. The protein sample was collected by centrifugation at 16,000 rpm for 20 minutes. A supernatant was collected, and the amount of a protein was measured by the Bradford assay. The conservation of the protein stability may be seen by subtracting a later-measured value from the initially-measured value. The Bradford assay was performed by mixing 40 μl of a Bio-Rad protein assay dye reagent concentrate produced by Biorad with 160 μl of a diluted protein for 5 minutes at 30° C., and an absorbance was measured at a wavelength of 595 nm using a microassay reader to deduce a result. As a standard material, 10 mg of bovine serum albumin (BSA) was used. That is, an absorbance value of the protein sample obtained in a calibration curve from the standard material was calculated, and a difference from the actual concentration of the protein may be calculated.
To confirm a type of a reaction product produced by the recombinant HcCel5H, 10% Avicel was used as a substrate, and a reaction was performed under conditions of a total reaction amount of 200 μl, 50° C., pH 6, and a 0.1M sodium citrate buffer. The reaction products was sampled with respect to time, and analyzed by (a) DNS and (b) thin liquid chromatography (TLC), respectively.
The DNS analysis was performed by the same method as in the examples. In the TLC analysis, a mixture of butanol:acetic acid:water at a ratio of 2:1:1 was used as a developing solution, and a TLC plate was covered with silica. After development, 10% sulfate-containing ethanol was sprayed and dried, and a color reagent was sprayed and dried again. Afterward, coloring was confirmed by applying heat.
To confirm a type of an oligosaccharide capable of being decomposed by the recombinant HcCel5H, 1% oligosaccharide was used as a substrate, and a reaction was performed under conditions of a total reaction amount of 20 μl, 50° C., pH 6, and a 0.1M sodium citrate buffer for 20 minutes. The resultant products were analyzed by thin liquid chromatography (TLC). An analysis method is the same as described in Experimental Example 6.
The high processivity of the cellulase, which was confirmed in
Determination of an enzyme kinetics constant in an experiment by the Michaelis-Menten equation was used since the Michaelis-Menten equation is a general method of obtaining the kinetic constant of an enzyme. Table 1 shows the Michaelis constant Km, the maximum velocity Vmax and the catalytic constant Kcat, obtained from the experiment. Table 1 shows precisely quantified and objectified data of actual reaction patterns between the recombinant cellulase and a substrate. An experimental method is as follows. In consideration of the initial reaction rate, various substrate concentrations from 1 to 4% (w/v) with respect to CMC were used, to which 0.1 nmol of the same recombinant cellulase was added to each sample, and a reaction time was 5 minutes. For Avicel, various substrate concentrations from 0.8 to 4% (w/v) were used, to which 0.5 nmol of the recombinant cellulase was added to each sample, and the reaction time was 2 hours. As a reaction solution, 200 μl of 50 mM sodium citrate (pH 6.0) was used and allowed to react at 50° C. From the double reciprocal plot called the Lineweaver Bulk plot using the experimental data obtained above, Km, Kcat, and Kcat values were calculated.
Rapid decomposition when the substrate having the lowest Michaelis constant Km, CMC, was used, demonstrates that the property of the endoglucanase is predominantly exhibited. However, unlike a general cellulase, the recombinant cellulase shows a relatively high activity even in the use of Avicel, and therefore it was also concluded that the recombinant cellulase also has the property of the exoglucanase. The great difference in decomposability depending on a substrate is caused by the property of a substrate such as much greater enzyme accessibility for the soluble substrate, not by the original property of the enzyme itself.
TABLE 1
Km
Vmax
Kcat
Substrate
Enzyme
(mg · ml−1)
(mg · ml−1 · min−1)
(min−1)
CMC
HcCel5H
21.34
1.67 × 10−1
9.76 × 10−1
Avicel
171.77
4.94 × 10−3
2.89 × 10−3
0.2 nmol of the recombinant cellulase HcCel5H was added to a substrate such as a 10 mg filter paper (Whatman No 1.). Here, a reaction solution was 200 μl of a 50 mM sodium citrate buffer (pH 6.0), and a reaction was carried out at 50° C. for 2 hours. To detect a soluble sugar, a supernatant was obtained, and reducing sugars were measured by DNS. To detect an insoluble sugar, a remaining filter paper was washed six times, and the insoluble sugar was detected by a BCA analysis. When the soluble sugar value is divided by the insoluble sugar value, the processivity of the recombinant cellulase may be measured. Accordingly, it can be noted that the cellulase having a high reactivity processively degrades substrates, and thus has a far higher content of the soluble sugar than the general cellulase, and as shown in Table 2, the recombinant enzyme has a high processive reactivity, for example, 5.78 (the processive reactivity of a general cellulase is 2 to 3).
TABLE 2
Level of processive
Enzyme
reactivity
% soluble sugar
% insoluble sugar
HcCel5H
5.78 ± 0.78
85.16
14.84
The activity of the recombinant HcCel5H was further confirmed using a viscometer. The viscometer used was DV-II+ Pro Extra (Brookfield, Middleboro, Mass., USA), and the activity was measured by detecting a viscosity value (Cp) decreased over time. A liquid solution was prepared by dissolving CMC in 25 ml of a 0.1 M sodium citrate buffer (pH 6.0) to have a concentration of 3% (w/v). 0.1 nmol of the recombinant HcCel5H was added to the liquid solution at room temperature, 30 ml of the liquid solution was poured in the viscometer, and a change in viscosity was observed while rotating the viscometer.
Lee, Hee Jin, Kim, Kyoung Heon, Kim, In Jung, Choi, In-Geol
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